Elsevier

Neuroscience

Volume 104, Issue 1, 10 April 2001, Pages 223-234
Neuroscience

Neuronal injury-induced expression and release of apolipoprotein E in mixed neuron/glia co-cultures: nuclear factor κB inhibitors reduce basal and lesion-induced secretion of apolipoprotein E

https://doi.org/10.1016/S0306-4522(01)00046-XGet rights and content

Abstract

In order to better delineate the intracellular signaling pathways underlying glial apolipoprotein E (apoE) expression and release, we have characterized an in vitro model of induction of glial apoE production induced by neuronal death. Exposure of mixed fetal cortical neuron/glia co-cultures to the neurotoxin N-methyl-d-aspartate results in increased apoE expression and release in a time- and concentration-dependent manner. Increased expression of apoE messenger RNA precedes the increase in intracellular apoE, followed by accumulation of the holoprotein in the culture medium. Neuronal injury induced by N-methyl-d-aspartate is accompanied by a reactive astrogliosis as measured by an increase in glial fibrillary acidic protein messenger RNA and protein at 48 and 72 h post-lesion, respectively. A similar microgliosis was observed using the microglial marker ED-1. Neuronal injury-induced glial apoE secretion is attenuated by the nuclear factor κB inhibitors, aspirin, Bay 11-7082 and MG-132, suggesting that this transcription factor is involved in both constitutive and induced glial apoE expression.

The present data show that up-regulation of apoE is an early event in the glial activation triggered by neurodegeneration in vitro and that activation of nuclear factor κB directly or indirectly mediates the increase in apoE expression.

Section snippets

Cell cultures

Mixed neuron/glia cultures were prepared from fetal 18-day-old Sprague–Dawley rat embryos (Harlan, Indianapolis, IN, USA). In brief, brain tissue was dissected on ice, and the cerebral cortices were freed of meninges and dissociated in 5 ml of 0.05% trypsin/0.53 mM EDTA for 15 min at 37°C. Trypsin was then neutralized by addition of DNase (200 U/ml) and minimum essential medium (MEM) containing 10% fetal bovine serum, and the cell suspension was centrifuged at 200g for 4 min. The pellet was

Characterization of the model: N-methyl-d-aspartate-induced neuronal death and glial proliferation

We first confirmed that exposure of mixed neuron/glia cultures to NMDA induced a concentration-dependent neuronal death, measured by LDH release. The neurotoxic effect of NMDA was maximal at 100 μM (15–25% of maximal cell death; Fig. 1A). Next, we studied whether NMDA-induced neuronal death was accompanied by glial proliferation. As seen in Fig. 1B, the number of proliferating cells in control wells was highest at days 1 and 2 and then declined. NMDA treatment did not induce significant

Discussion

The APOE4 allele confers a high susceptibility for late-onset and sporadic AD and a poor outcome after head trauma promoting amyloid deposition, therefore enhancing the risk for AD.25., 43., 54., 55. However, APOE4 is neither necessary nor sufficient for developing AD, since this risk factor accounts for only 30–50% of the late-onset cases.18 Environmental or other biological factors may influence the onset of the disease. For instance, apoE expression, independent of genotype, could be a

Acknowledgements

J.S. is a recipient of a grant from the Spanish Ministry of Education. We gratefully thank Dr Neil DeLapp for help in western and northern quantitation, and Pamela Edmonds for excellent editorial work.

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    V. Petegnief and J. Saura contributed equally to this work.

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